Field of the Invention
The present invention relates to a method for determining the concentration of particles dispersed and moving randomly in a sample solution and bound to particles for separation and recovery by solid-liquid separation treatment using an optical system capable of detecting light from a microregion in the solution, such as an optical system of a confocal microscope and multi-photon microscope.
The present application claims priority on the basis of Japanese Patent Application No. 2011-187600, filed in Japan on Aug. 30, 2011, the contents of which are incorporated herein by reference. The present application is a U.S. continuation application based on the PCT International Patent Application, PCT/JP2012/066576, filed on Jun. 28, 2012; the contents of which are incorporated herein by reference.
Description of the Related Art
Due to progress made in the field of optical measurement technology in recent years, it has become possible to detect and measure feint light at the level of a single photon or single fluorescent molecule using the optical system of a confocal microscope and ultra-high-sensitivity photodetection technology capable of performing photon counting (detecting individual photons). Therefore, various devices or methods have been proposed that detect interactions between molecules such as biomolecules or coupling and dissociation reactions between molecules using such feint light measurement technology. For example, in fluorescence correlation spectroscopy (FCS: see, for example, Japanese Unexamined Patent Application, First Publication No. 2005-098876; Japanese Unexamined Patent Application, First Publication No. 2008-292371; Japanese Unexamined Patent Application, First Publication No. 2009-281831; Kinjo, M., Proteins, Nucleic Acids and Enzymes, 1999, Vol. 44, No. 9, pp. 1431-1438; Meyer-Almes, Fluorescence Correlation Spectroscopy, R. Rigler, ed., Springer, Berlin, 2000, pp. 204-224; and Katoh, N., et al., Gene and Medicine, 2002, Vol. 6, No. 2, pp. 271-277), fluorescence intensity is measured from fluorescent molecules or fluorescent-labeled molecules (such as fluorescent molecules) entering and leaving a microregion (a region where laser light of a microscope is focused; referred to as confocal volume) in a sample solution using the optical system of a laser confocal microscope and photon counting technology. Information such as the speed of movement or the size or concentration of fluorescent molecules and the like is acquired, or various phenomena in the manner of changes in molecular structure or size, molecule coupling and dissociation reactions or dispersion and aggregation are detected, based on the average retention time (transitional diffusion time) of fluorescent molecules and the like in the microregion and the average value of the number of molecules remaining therein determined from the value of an autocorrelation function of the measured fluorescence intensity. In addition, in fluorescence intensity distribution analysis (FIDA: see, for example, Japanese Patent (Granted) Publication No. 4023523) and photon counting histograms (PCH: see, for example, International Publication No. WO 2008-080417), a histogram is generated of the fluorescence intensity of fluorescent molecules and the like entering and leaving a measured confocal volume in the same manner as FCS. By fitting a statistical model formula to the distribution of that histogram, the average value of the characteristic brightness of the fluorescent molecules and the like and the average value of the number of molecules remaining in the confocal volume are calculated, and changes in molecular structure and size, coupling and/or dissociation, or dispersion or aggregation and the like are then estimated based on this information. Moreover, Japanese Unexamined Patent Application, First Publication No. 2007-20565 and Japanese Unexamined Patent Application, First Publication No. 2008-116440 propose a method for detecting a fluorescent substance based on the time lapse of a fluorescent signal of a sample solution measured using the optical system of a confocal microscope. Japanese Unexamined Patent Application, First Publication No. H04-337446 proposes a signal arithmetic processing technology for detecting the presence of fluorescent fine particles in a flow or on a substrate by measuring feint light from fluorescent fine particles that have passed through a flow cytometer or fluorescent fine particles immobilized on a substrate using photon counting technology.
In particular, according to methods using microregion fluorescence measurement technology using the optical system of a confocal microscope and photon counting technology in the manner of FCS or FIDA and the like, the sample required for measurement is only required to be of an extremely low concentration and extremely small amount in comparison with that used in the past (since the amount used for a single measurement is roughly only several tens of microliters) and measurement time is shortened considerably (measurement of a duration on the order of several seconds for a single measurement is repeated several times). Thus, these technologies are expected to be utilized as powerful tools that make it possible to carry out experimentation or testing less expensively and faster in comparison with conventional biochemical methods in the case of performing analyses on scarce or expensive samples frequently used in fields such as medical or biochemical research and development, or in the case of a large number of specimens such as when clinically diagnosing diseases or screening physiologically active substances.